DISTRIBUTION STATEMENT A - PDF document

1 '" SDTIC EI"ECTE DISTRIBUTION STATEMENT
'" SDTIC EI"ECTE DISTRIBUTION STATEMENT A Approved for public relecise t Distribution Unlimited Plasmids, Pasteur, and Anthrax into healthy animals (18), and 15 years later the anthrax bacillus was observed microscopically by Dela- fond (2). During the 1850s and Perry Mikesell, Bruce E. Ivins, Joseph D. Ristroph, Michael H. Vodkin, 1860s, numerous studies established Thomas M. Dreier, and Stephen H. Leppla that Wi) blood forom anthrax-infect-d Divisions of Bacteriology and Pathology, U.S. Army Medical Research animals contained large, nonmotile Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701 bacilli, (ii) inoculation of healthy animals with blood or tissue con- Few microbial pathogens have been a gradual decline in the inci- taining these bacilli engendered the had as great an impact oil the devel- dence of anthrax in humans in the disease, and (iii) the "malignant "opment of the science of medical United States. This is attributed to pustule" in humans contained bacil- bacteriology as Bacillus anthracis. three major factors: (i) the use of a li similar to those found in animals � This organism is the etiological vaccine for individuals deemed at with anthrax. Yet it was not until Lagent of anthrax (Greek; "coal, car- risk, (ii) better working conditions 1877 that Robert Koch conclusively Z buncle, pustule"), a historical dis- for those exposed to wool, hides, and proved that there was a causal rela- c.ease of considerable economic im- animal products, and (iii) less expo- tionship between the large nonmo- portance. Anthrax-like infections sure to imported contaminated ani- tile bacilli and the disease of an- LLjare described in ancient literature, mal products (2). thrax (18). In so doing Koch became _ including the biblical Book of Exo- the first to demonstrate that a spe- dus, which describes the disease as cific bacterium was responsible for a the fifth plague of Egypt. Anthrix Wstory of Anthrax Research specific disease. This work resulted CZepizootics were responsible for enor- Although anthrax dates back in the formulation of Koch's famous Smous domestic livestock losses in more than 2,000 years, it was not postulates, which established the Europe from the seventeenth recognized as a disease until the framework of theory and practice for through the nineteenth century. eighteenth century. Maret in 1152 the development of the science of The first infections in animals in the and Fourn-ier in 1769 both~described medical microbiology. United States were documented in the "malignant pustule" in humans, In France in the late 1870s an- the Louisiana Territory in the early and Chabert in 1780 described the thrax was a severe disease that de- Seighteenth century. The disease disease in animals (19). However, no stroyed flocks of sheep throughout spread throughout the South and connection between the two diseases the French countryside. This de- Northeast, and in the 1820s the first was made for the next 80 years. In struction influenced Louis Pasteur human cases of anthrax in this 1853, Bartheldmy demonstrated to direct his attention to the study of country were reported in Kentucky. transmission of the disease by inocu- anthrax. Pasteur had just developed Since the early 19G0s, there has lating blood from infected animals a regimen for heat attenuation of 320 ASM News "tS ohe causative agent of chicken chol- considered innocuous, the PA corn- Plasmid Studies era, and he used a similar approach ponent has been reported to effect in his studies of virulent anthrax transient alterations in neural and Cell-free supernatant fluids from bacilli (18). Cultures of the organism cardiovascular function in chal- broth cultures of two nonencapsulat- grown at elevated temperatures lenged hosts (17), whereas neither ed toxigenic strains of B. anthracis were shown to be decreased in viru- EF nor LF alone is biologically ac- (Sterne and V770-NP1-R) and an .lence. In historic field trials at Pouil- tive. However, PA in combination encapsulated, toxigenic strain (Vol- ly-le-Fort in 1881, Pasteur showed with EF or LF produces localized lum 1B) grown in a chemically de- that these heat-attenuated orga- edema or death, respectively, in ex- fined medium (12) at 37 (optimal) nisms were capable of producing im- perimental animals (8). Many inves- and 42.5°C (heat-treated) have been munity against later challenge with tigators have tried to elucidate the assayed for lethal toxin (1, 5) and "virulent strains of the bacillus in role of the holotoxin in the disease edema production (13). Both activi- animals. Pasteur is therefore given process, in the belief that under- ties were readily detectable in par- credit for developing the first vac- standing the toxin's mechanism of ent strain superratants, whereas cine effective in the prevention of action would aid in developing a neither activity was found in culture anthrax

2 (18). safe, effective, and long-lasting
(18). safe, effective, and long-lasting hu- supernatants from the heat-treated Mention must be made, however, man vaccine, strains. Furthermore, loss of viru- of W. S. Greenfield, Professor Super- lence was noted in cells subjected to intendent of the Brown Animal San- A licensed vaccine against an- heat treatment. awry Institution in England (1878- thrax, which appears to afford some Single plasmid species that dif- 81). Greenfield, like Pasteur, antici- protection from the disease, is cur- fered in molecular weight were de- pated the results of growing cultures rently available for human use (3). tected in each parental B. anthracis of the anthrax bacillus at high tern- The vaccine contains alum-precip- strain. Plasmid DNA, however, perature and actually performed ex- itated supernatant material from could not be detected in the heat- periments similar to Pasteur's some fermenter cultures of an avirulent treated isolates, suggesting a corre- months before the trials at Pouilly- B. anthracis strain (V770-NP1-R). lation between the presence of plas- le-Fort (16). However, recognition Although an acceptable human vac- mid elements and the ability of the for his scientific achievements was cine now exists, there are reasons to organism to produce the toxin or not forthcoming, partly owing to fi- believe it can be improved. Fish et regulate its production. Definitive "nancial and political considerations al. (4) reported that purified PA is a evidence of plasmid-associated toxin of the time. Therefore, Greenfield much more effective protective im- production was obtained by trans- may actually deserve the credit for munogen than crude PA, yet the forming heat-treated cells with plas- developing the first anthrax vaccine, current vaccine consists of a crude mid DNA purified from the parent a point of contention perhaps best supernatant precipitate consisting strain. Biological acti-rities in the left to the medical historians and primarily of PA. Mahlandt et al. (9) sunernatant were restored, and S presented here only for the sake of reported that LF was a highly effec- plasmid DNA similar in molecular historical accuracy. tive protective immunogen, protect- weight to that of the parent strain ing rats against both toxin and spore was isolated from cultures of the challenge and guinea pigs against transformants (10). The Anthrax Toxin and Vaccine spore challenge, and Stanley and The locus of the PA structural "Development Smith (15) reported that the combi- gene was identified by recombinant nation of EF and PA afforded better DNA techniques (M. H. Vodkin and In the 100 years that followed protection than PA alone. There is S. H. Leppla, submitted for publica- these landmark studies, much was also strong evidence that immunity tion). Restriction fragments of plas- learned about the physiology and to anthrax is dramatically increased mid DNA purified from the Sterne pathobiology of this unique orga- by administering first the PA vac- strain of B. anthracis were cloned nism. It is now generally accepted cine and then a booster of live atten- into an Escherichia coli K-12 that to be fully virulent, B. anthracis uated bacteria (6). pBR322 host vector system. Two strains must possess both a polyglu- clones were identified as producing tamic acid capsule and a protein The goal of ongoing research a protein which reacted with anti- exotoxin composed of three factors. with the anthrax bacillus is to pro- body to PA and was indistinguish- The production of this toxin in vivo duce a more efficacious human vac- able from purified PA when ana- "was suggested by the work of Cro- cine, with the PA moiety used as the ly-ed by gel electrophoresis and martie et al. in 1947 (3a), and its central element. Modern techniques Western blotting techniques. A bio- presence was conclusively proven in in molecular biology and recombi- logical assay showed that this pro- 1955 (13). By the early 1960s it was nant DNA technology have been tein was a functional PA gene prod- recognized as a complex toxin or used toward this end, with the hope uct (7). Stoxic mixture composed of three pro- that such studies might provide a Through recombinant DNA tein factors: edema factor (EF), pro- molecular explanation of the suc- technology, a strain of E. coli was tective antigen (PA), and lethal fac- cesses achieved by Pasteur and thus constructed which contained a tor (LF) (1, 14). Although generally Greenfield a century ag'). subgenomic fragment of plasmid VOL. 49, NO. 7, 1983 321 *'/ "DNA from B. anthracis. This recom- to continue work initiated more eukaryotic cells. Proc. Natl. Acad. Sci. binant molecule contained the struc- than a century ago. Many individual U.S.A. 79:3162-3166. tural gene(s) for PA. This protein advances -electron microscopy, the 8. Lincoln, R. E., and 1). C. Fish. 1970. bddtuAnthrax toxin, p. 361-414. In T. C. Mon- can now be harvested from non- de

3 coding of the structure, compo- tie, S.
coding of the structure, compo- tie, S. Kadis, and S. J. Ajl (ed.), Microbial pathogenic bacterial cultures free of nents, and behavior of DNA, the toxins. Academic Press, Inc., New York. the other two components and in- development of genetic engineering, 9. Mahlandt. B. G., F. Klein. R. E. Lincoln, deed free of any other contaminat- and sophisticated assay techniques B. W. Haines, W. 1. Jones, Jr., and R. H. ing Bacillus proteins. Future ma- -were required before a simple Friedman. 1966. Immunological studies of anthrax. IV. Evaluation of the immu- nipulations to specifically enhance answer to Pasteur's simple question nogenicity of three components of an- PA production can now be planned. could be found. thrax toxin. J. Immunol. 96:727-733. These accomplishment,, have led to 10. Mikesell, P.. B. E. Ivins, J. D. Ristioph, the threshold of the primary objec- Literature Cited and T. D. Dreier. 1983. Evidence for tive -the development of a safe, plasmid-mediated toxin production in more efficacious human anthrax 1. Beall, F.A., M. J. Taylor, and C. B. Bacillus anthracis. Infect. Immun. Thorne. 1961. Rapid lethal effect in rats 39:371-376. vaccine. of a third component found upon fraction- 11. Pasteur, L. 1881. De l'attknuation des During the course of his studies ating the toxin of Bacillus anthracis. J. virus et de leur retour A la virulence. C. on anthrax, Pasteur asked how viru- Bacteriol. 83:1274-1280. R. Acad. Sci. Agric. Bulg. 92:429-435. lence could be lost during 8 days at 2. Brachman, P. S. 1970. Anthrax. Ann. 12. Ristroph, J. D., and _P E. [vins. 1983. N.Y. Acad. Sci. 174:577-582. Elaboration of Bacillus anthracis anti- 43°C (11). A reasonable answer to 3. Brachman, P.S., H. Gold, S. A. Plotkin, gens in a new, defined culture medium. this question can now be given and F. R. Fekety, M. Werrin, and N. R. In- Infect. Inmun. 39:483-486. can also explain the observations graham. 1962. Field evaluation of a hu- 13. Smith, H.. J. Keppie, and J. L. Stanley. made by Pasteur and Greenfield. In man anthrax vaccine. Am. J. Public 1955. The chemical basis of the virulence Health 52:632-645. of Bacillus anthiacis. V. The specific tox- addition to the studies already de- 3a. Cromartie, W. J., D. W. Watson, W. L. in produced by B. anthracis in vivo. Br. J. scribed, two Pasteur vaccine strains Bloom, and R. J. Heckly. 1947. Studies Exp. Pathol. 36:323-335. of B. anthracis (ATCC 4229 and on infection with Bacillus anthraczs. II. 14. Stanley, J. L., and H. Smith. 1961. Puri- ATCC 6602) were examined for bio- The immunological and tissue damaging fication of factor I and recognition of a logical and serological toxin activity properties of extracts prepared from the third factor of the anthrax toxin. J. GCn. lesions of B. anthracis infection. J. Infect. Microbiol. 26:49-66. and for the presence of plasmids. Dis. 80:14-27. 15. Stanley, J. L., and H. Smith. 1963. The These strains contained no detect- 4. Fish, D. C., B. G. Mahlandt, J. P. three factors of anthrax toxin: their im- able plasmid elements. Further- Dobbs, and R. E. Lincoln. 1968. Purifi- munogenicity and lack of demonstrable more, they resembled the heat- cation and properties of in vitro-pro- enzymic activity. J. Gen. Microbiol. treated strains in lethal toxin, duced anthrax toxin components. J. Bac- 31:329-337. Steriol. 95:907-918. 16. Tigertt, W. D. 1980. Anthrax. J. Hyg. edema-producing, and serological 5. Haines, B. W., F. Klein, and R. E. Lin- 85:415-420. activities. It is likely, therefore, that coin. '1965. Quantitative assay for crude 17. Vick. J. A., IR E. Lincoln, F. Kleivi, B. G. Pasteur and Greenfield attenuated anthrax toxin.. J. Bacteriol. 89:74-83. Mahlandt, J. S. Walker, and D. C. Fish. the anthrax bacillus by using heat 6. Klein, F., I. H. DeArmon, Jr., B.G. 1968. Neurological and physiological re- Mahlandt, R. E. Lincoln, and A. L. Fer- sponses of the primate to anthrax toxin. to cure the strains of temperature- nelius. 1962. Immunological studies of J. Infect. Dis. 118:85-96. sensitive plasmid elements encoding anthrax. II. Levels of immunity against 18. Wilson, G. S., and A. Miles. 1975. Topley one if not all of the toxin proteins. Bacillus anthocis obtained with protec- and Wilson's principles of bacteriology, Science is the process of answer- tive antigen and live vaccine. J. Immu- virology and immunity, p. 2208-2224. nol. 88:15-19. The Williams & Wilkins Co., Baltimore. ing seemingly simple questions, 7. Leppla, S. H. 1982. Anthrax toxin edema 19. Wistreich, G. A., and M. D. Lehman. such as that posed by Pasteur. It is factor: a bacterial adenylate cyclase that 1973. Microbiology and human disease, indeed rare to have the opportunity increases cyclic AMP concentrations in p. 493-497. Glencoe Press, New York. Accession fti NTIS GRA&I DTIC TAB Unannounced Justification "By Distribution/ AvailabilityCod SAvail and,/Aw Dist Special *322 AlASM News 'SLA i *.